Cells in the body respond to extracellular stimuli, that are both biochemical and mechanical in nature (Vandenburgh, Am. J. Physiol 262:R350-355, 1992; Buckley, Bone Miner. 4:225-236, 1988; Brunette, Cell Science, 69:35-45, 1984; Harris, 3. Biomech Engineering, 106:19-24, 1984). Both endothelium and muscle respond dynamically to mechanical stimuli and serve as signal transduction interfaces. Although a much focused research topic in cell physiology, there are some fundamental issues in experimental set-up of muscle cell cultures which have not been adequately addressed.
Mechanobiological studies usually involve statically strained membranes upon which cell monolayers are grown. However, such in vitro approaches are ineffective at providing a good indication of cell function in vivo for a number of reasons. Firstly, these cell culture systems produce significant detachment between the membrane that is being stretched and the overlying substrata. Secondly, unlike the complex three-dimensional force effects seen in vivo, the traditional in vitro culture systems forces are transmitted in only one direction. Furthermore, the complex three-dimensional arrangement of myocytes, and in particular, cardiac myocytes as found in vivo, is usually lacking in the in vitro models. Therefore, in understanding the role of mechanical stimuli upon cell functional processes in culture, it would be beneficial to provide an appropriate membrane or matrix that will more closely mimic the in vivo cellular arrangement.
An example of this can be seen in studies examining the effects of stretch on cardiac gene regulation. In such experiments, myocytes, usually rat cardiac myocytes, are grown in monolayer culture upon silicone and subjected to external mechanical stress. There have been studies of cardiac myocytes, in which the rate of protein synthesis for non-aligned cells has been measured using silicone membranes that used collagen to keep cells attached. (Terracio et al., In Vitro Cellular and Developmental Biology, Vol. 24, 1988; Sharp et al., Circ. Res. 73: 172-183, 1993; Am. J. Physiol, 42: H546-H556, 1997). However, even though myocytes do adhere to collagen quite well in static culture, there are still significant problems with detachment of the collagen layer from the silicone substrate upon repeated mechanical deformation. It is not surprising that this occurs, especially since it is well established that proteins and cells do not exhibit good adherence to smooth, low surface energy materials such as silicone.
To date, primary neonatal cultures have been the mainstay in the study of myocyte function since contractile cardiac cell lines are not available. However, when it comes to the study of the contractile function and processes of assembly primary neonatal cells are woefully inadequate since they generally have very few functioning myofibrils. Contractile activity is clearly an important signal in regulation of myocyte cell shape that leads, in turn, to remodeling the shape and function of the whole heart. Unfortunately, most adult and neonatal myocyte culture systems display little or no contractile activity.
Thus, there is a need for phenotypically normal myocytes that can be manipulated experimentally. Furthermore there is a need to develop a culture substrata that allows cells to adhere and remain adhered during the application of mechanical and other force.